The present invention is in the field of nucleic acid amplification, and specifically in the area of reducing amplification artifacts in nucleic acid amplification reactions.
Numerous nucleic acid amplification techniques have been devised, including strand displacement cascade amplification (SDCA)(referred to herein as exponential rolling circle amplification (ERCA)) and rolling circle amplification (RCA)(U.S. Pat. No. 5,854,033; PCT Application No. WO 97/19193; Lizardi et at., Nature Genetics 19(3):225–232 (1998)); multiple displacement amplification (MDA)(PCT Application WO 99/18241); strand displacement amplification (SDA)(Walker et al., Nucleic Acids Research 20:1691–1696 (1992), Walker et al., Proc. Natl. Acad. Sci. USA 89:392–396 (1992)); polymerase chain reaction (PCR) and other exponential amplification techniques involving thermal cycling, self-sustained sequence replication (3SR), nucleic acid sequence based amplification (NASBA), and amplification with Qβ replicase (Birkenmeyer and Mushahwar, J. Virological Methods 35:117–126 (1991); Landegren, Trends Genetics 9:199–202 (1993)); and various linear amplification techniques involving thermal cycling such as cycle sequencing (Craxton et al., Methods Companion Methods in Enzymology 3:20–26 (1991)).
Artifacts—that is, unwanted, unexpected, or non-specific nucleic acid molecules—have been observed in almost all nucleic acid amplification reactions. For example, Stump et al., Nucleic Acids Research 27:4642–4648 (1999), describes nucleic acid artifacts resulting from an illegitimate PCR process during cycle sequencing. Stump et al. suggests a way to avoid such artifacts by using certain primers that cannot be fully replicated. Watson, Amplifications, 5–6 (1989), and Ferrie et al., Am. J. Hum. Genet. 51:251–262 (1992), describe formation of primer-dimer artifacts during PCR. Brownie et al., Nucleic Acids Research 25:3235–3241 (1997), suggests a way to avoid primer dimer formation during PCR by adding tails at the 5′ end of the PCR primers that results in formation of a non-replicable structure if primer-dimer formation is initiated. Other forms of artifacts can occur in other types of nucleic acid amplification techniques.
Therefore, it is an object of the present invention to provide a method of reducing, preventing, or eliminating artifacts in nucleic acid amplification reactions.
It is another object of the present invention to provide oligonucleotides that, when used in a nucleic acid amplification reaction, can reduce, prevent, or eliminate artifacts in the nucleic acid amplification reaction.
It is another object of the present invention to provide kits for nucleic acid amplification that can reduce, prevent, or eliminate artifacts in the nucleic acid amplification reaction.